Isothiocyanate-functionalized RGD peptides for tailoring cell-adhesive surface patterns

With the advances made in surface patterning by micro- and nanotechnology, alternative methods to immobilize biomolecules for different purposes are highly desired. RGD peptides are commonly used to create cell-attractive surfaces for cell-biological and also medical applications. We have developed a fast, one-step method to bind RGD peptides covalently to surfaces by thiourea formation, which can be applied to structured and unstructured materials. RGD peptides were fused to an isothiocyanate anchor during synthesis and directly immobilized on amino-terminated surfaces. The spreading behavior of fibroblasts and the formation of focal contacts served to prove the applicability of the coupling method. Two different linear peptides and one cyclic peptide were compared. All the peptides induced spreading behavior and the formation of focal contacts in murine fibroblasts. Adhesion was specific as cells neither recognized the corresponding negative control peptides nor spread in the presence of soluble H-RGDS-OH peptide. We successfully applied our coupling method to functionalize surface patterns created by microcontact printing (μCP) and chemical etching. Cells recognize areas selectively coated with RGD-containing peptides, proliferate and maintain this preference during long-term cultivation. Our method significantly facilitates surface modification with any kind of peptide – even for the preparation of peptide-functionalized small surface areas.     

Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces

The demand for orthopedic and dental implants will continue to grow, and for these applications, titanium and its alloys have been used extensively. While these implants have achieved high success rates, two major complications may be encountered: the lack of bone tissue integration and implant-centered infection. The surface of the implant, through its interactions with proteins, bacteria and tissue cells, plays a determining role in the success or failure of the implant. Ideally, to enhance the success of implants, their surfaces should inhibit bacterial colonization and concomitantly promote osteoblast functions. In this article, we discuss strategies for tailoring implant surfaces by exploiting the differences in the response of bacteria and osteoblasts to proteins and surface structures. Nevertheless, limitations still exist in the quest for an ideal implant surface. Further advances in this field will require concurrent development in surface modification techniques and a better understanding of the complex and highly inter-related events occurring at the implant surface after implantation.     

Micropatterned surfaces of PDMS as growth templates for HEK 293 cells

In this paper the easy and reliable preparation of precise micropatterns on PDMS surfaces is described and the growth of HEK 293 cells on those patterns during culture over several days is examined. The first patterning approach described is based on soft-lithography and polyelectrolyte multilayer deposition. Two different soft-lithographic techniques are employed for creating surface patterns of PAH, PSS, untreated and oxidized PDMS. The growth behavior of HEK 293 cells is investigated on all the dual combinations of the four surfaces, and decreasing preference of the cells for the surfaces in the order PAH (–NH₂) > ox-PDMS (–OH) >> PSS (–SO₃ ⁻) > PDMS (–CH₃) is revealed. As the second patterning approach a method is introduced, which allows the deposition of gel droplets in a microarray format utilizing differences in the surface wettability. This concept is new and expected to be very useful for various applications. Finally, a speculative explanation for the different cell spreading behavior is provided considering the interplay between individual cell–surface interactions and a permanent cell tractional force.     

Polyelectrolyte/silver nanocomposite multilayer films as multifunctional thin film platforms for remote activated protein and drug delivery

We demonstrate a nanoparticle loading protocol to develop a transparent, multifunctional polyelectrolyte multilayer film for externally activated drug and protein delivery. The composite film was designed by alternate adsorption of poly(allylamine hydrochloride) (PAH) and dextran sulfate (DS) on a glass substrate followed by nanoparticle synthesis through a polyol reduction method. The films showed a uniform distribution of spherical silver nanoparticles with an average diameter of 50 ± 20 nm, which increased to 80 ± 20 nm when the AgNO₃ concentration was increased from 25 to 50 mM. The porous and supramolecular structure of the polyelectrolyte multilayer film was used to immobilize ciprofloxacin hydrochloride (CH) and bovine serum albumin (BSA) within the polymeric network of the film. When exposed to external triggers such as ultrasonication and laser light the loaded films were ruptured and released the loaded BSA and CH. The release of CH is faster than that of BSA due to a higher diffusion rate. Circular dichroism measurements confirmed that there was no significant change in the conformation of released BSA in comparison with native BSA. The fabricated films showed significant antibacterial activity against the bacterial pathogen Staphylococcus aureus. Applications envisioned for such drug-loaded films include drug and vaccine delivery through the transdermal route, antimicrobial or anti-inflammatory coatings on implants and drug-releasing coatings for stents.     

LPAM-1 (integrin α4β7)-ligand binding: overlapping binding sites recognizing VCAM-1, MAdCAM-1 and CS-1 are blocked by fibrinogen,a fibronectin-like polymer and RGD-like cyclic peptides

The α4 integrin LPAM-1 (α4β7) mediates lymphocyte attachment within the extracellular matrix (ECM) by adhering to the connecting segment (CS)-1 site of fibronectin (FN). Here we reveal that very late antigen (VLA)-4⁻ LPAM-1⁺ T cell lymphoma TK-1 cells bind via LPAM-1 to multiple copies of the RGD sequence engineered within an FN-like polymer. Further, the small conformationally restrained RGD-like cyclic peptides 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys and Arg-Cys-Asp-thioproline-Cys inhibit the adhesion of TK-1 cells to immobilized CS-1 peptide, and to endothelial counterreceptors for LPAM-1, namely mucosal addressin cell adhesion molecule (MAdCAM)-1 and vascular cell adhesion molecule (VCAM)-1. Spontaneous adhesion of the VLA-4⁻ LPAM-1⁺ B lymphoma cell line RPMI 8866 to CS-1 was likewise inhibited, confirming a previously undocumented ability of LPAM-1 to recognize the RGD tripeptide. The RGD-binding site in LPAM-1 either overlaps or is identical to sites required for interaction with MAdCAM-1, VCAM-1, and the CS-1. The binding of LPAM-1 and VLA-4 to RGD-containing ligands may have relevance in vivo given that fibrinogen at physiological concentrations is able to partially block the binding of TK-1 cells to MAdCAM-1. Hence fibrinogen and other vascular RGD-containing proteins may have mild anti-inflammatory activity required for maintaining effective homeostasis, analogous to the anti-thrombogenic activity of the vascular endothelium.     

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.     

Polyelectrolyte multilayer films with pegylated polypeptides as a new type of anti-microbial protection for biomaterials

Adhesion of bacteria at the surface of implanted materials is the first step in microbial infection, leading to post-surgical complications. In order to reduce this adhesion, we show that poly(L-lysine)/poly(L-glutamic acid) (PLL/PGA) multilayers ending by several PLL/PGA-g-PEG bilayers can be used, PGA-g-PEG corresponding to PGA grafted by poly(ethylene glycol). Streaming potential and quartz crystal microbalance-dissipation measurements were used to characterize the buildup of these films. The multilayer films terminated by PGA and PGA-g-PEG were found to adsorb an extremely small amount of serum proteins as compared to a bare silica surface but the PGA ending films do not reduce bacterial adhesion. On the other hand, the adhesion of Escherichia coli bacteria is reduced by 72% on films ending by one (PLL/PGA-g-PEG) bilayer and by 92% for films ending by three (PLL/PGA-g-PEG) bilayers compared to bare substrate. Thus, our results show the ability of PGA-g-PEG to be inserted into multilayer films and to drastically reduce both protein adsorption and bacterial adhesion. This kind of anti-adhesive films represents a new and very simple method to coat any type of biomaterials for protection against bacterial adhesion and therefore limiting its pathological consequences.     

A spatial patternable macroporous hydrogel with cell-affinity domains to enhance cell spreading and differentiation

Cell adhesion and spreading are two essential factors for anchorage-dependent cells such as osteocytes. An adhesive macroporous hydrogel system, in which cell-affinitive domains and sufficient cytoskeleton reorganization space were simultaneously constructed, was proposed in this report to support cell adhesion and spreading, respectively, and facilitate cell differentiation and function establishment eventually. The adhesive macroporous alginate hydrogel was developed by RGD peptide graft and gelatin microspheres hybridization to generate cellular adhesion sites and highly interconnected macropores. The successful stretched morphology and enhanced osteogenic differentiation of MG-63 cells in this modified alginate hydrogel showed clearly the feasibility that cell function may be effectively facilitated. Besides, this hydrogel model can be further applied to construct complex micropatterned structure, such as individual microgels in shapes of circle, square, cross and ring, and osteon-like structure containing both osteogenic and vascularized area generated by a double-ring assembly. These results should provide this adhesive macroporous photocrosslinkable hydrogel system as potential three-dimensional scaffolds for guiding tissue formation, especially for the bioengineering of tissues that have multiple cell types and require precisely defined cell–cell and cell–substrate interactions.     

中药复方补肾活血液对成骨细胞影响的实验研究

目的:观察中药复方补肾活血液对体外培养新生SD大鼠成骨细胞增殖、分化、矿化的影响。方法:采用二次酶消化法从新生SD大鼠颅骨获取成骨细胞进行培养, 取第3代成骨细胞为实验模型, 将其分为4组, 在各组中分别加入双蒸馏水(对照组), 低(20mg/L)、中(40mg/L)、高(80mg/L)浓度的补肾活血中药提取液。用MTT法(波长570nm处A值表示)测定细胞增殖功能;用对硝基苯基磷酸二钠(PNPP)法测定碱性磷酸酶(ALP)活性;用放免分析方法测定细胞培养液中的骨钙素(BGP)含量;用茜素红染色法, 在40倍光镜下作矿化结节计数。结果:中药中、高浓度组的A值在24h、48h、72h时明显高于对照组(P＜0.01), 且与药物浓度呈剂量依赖性;低浓度组的A值在72h时点明显高于对照组(P＜0.05), 在24h、48h时点与对照组相比无显著差异(P＞0.05)。不同浓度给药组培养48h、72h、96h后, ALP(除低浓度组培养48h外)、BGP均呈不同程度增加(P＜0.01)。在中、高浓度组矿化结节数量/视野, 显著多于对照组(P＜0.01), 低浓度组与对照组比较无显著差异(P＞0.05)。结论:补肾活血液具有促进体外培养的新生SD大鼠成骨细胞增殖、分化及矿化作用, 这种作用呈剂量相关性。     

Cell adhesion, spreading, and proliferation on surface functionalized with RGD nanopillar arrays

In this paper, a method was introduced for the fabrication of vertically and spatially-controlled peptide nanostructures that enhance cell adhesion, proliferation, spreading on artificial surfaces. The RGD nanostructures with different heights were fabricated on gold surfaces by self-assembly technique through a nanoporous alumina mask composed of nanoscale-controlled pores. Pore diameter and spatial distribution were controlled by manipulating the pore widening time at a constant voltage during the mask fabrication process. Two-dimensional RGD nanodot, three-dimensional RGD nanorod, and RGD nanopillar arrays were carried out using various concentrations of RGD peptide solution, self-assembly times, and pore sizes, which were 74 nm, 63 nm, and 43 nm in diameter, respectively. The fabricated RGD nanodot, nanorod, and nanopillar arrays were utilized as a cell adhesion layer to evaluate the cell adhesion force, adhesion speed, spreading assay, and phosphorylation of cofilin protein in PC12, HeLa, and HEK293T normal cells. Among the three different nanostructures, RGD nanopillar arrays were found to be suitable for cellular attachment, spreading, and proliferation due to the proper arrangement of the RGD motif, which mimics in vivo conditions. Hence, our newly fabricated RGD nanostructured array can be successfully applied as a bio-platform for improving cellular functions and in in vitro tissue engineering.     

Mimicking the extracellular matrix with functionalized,metal-assembled collagen peptide scaffolds

Natural and synthetic three-dimensional (3-D) scaffolds that mimic the microenvironment of the extracellular matrix (ECM), with growth factor storage/release and the display of cell adhesion signals, offer numerous advantages for regenerative medicine and in vitro morphogenesis and oncogenesis modeling. Here we report the design of collagen mimetic peptides (CMPs) that assemble into a highly crosslinked 3-D matrix in response to metal ion stimuli, that may be functionalized with His-tagged cargoes, such as green fluorescent protein (GFP-His₈) and human epidermal growth factor (hEGF-His₆). The bound hEGF-His₆ was found to gradually release from the matrix in vitro and induce cell proliferation in the EGF-dependent cell line MCF10A. The additional incorporation of a cell adhesion sequence (RGDS) at the N-terminus of the CMP creates an environment that facilitated the organization of matrix-encapsulated MCF10A cells into spheroid structures, thus mimicking the ECM environment.     

Surface functionalization of titanium with hyaluronic acid/chitosan polyelectrolyte multilayers and RGD for promoting osteoblast functions and inhibiting bacterial adhesion

Titanium (Ti) and its alloys are used extensively in orthopedic implants due to their excellent biocompatibility and mechanical properties. However, titanium-based implant materials have specific complications associated with their applications, such as the loosening of implant-host interface owing to unsatisfactory cell adhesion and the susceptibility of the implants to bacterial infections. Hence, a surface which displays selective biointeractivity, i.e. enhancing beneficial host cell responses but inhibiting pathogenic microbial adhesion, would be highly desirable. This present study aims to improve biocompatibility and confer long-lasting antibacterial properties on Ti via polyelectrolyte multilayers (PEMs) of hyaluronic acid (HA) and chitosan (CH), coupled with surface-immobilized cell-adhesive arginine-glycine-aspartic acid (RGD) peptide. The HA/CH PEM-functionalized Ti is highly effective as an antibacterial surface but the adhesion of bone cells (osteoblasts) is poorer than on pristine Ti. With additional immobilized RGD moieties, the osteoblast adhesion can be significantly improved. The density of the surface-immobilized RGD peptide has a significant effect on osteoblast proliferation and alkaline phosphatase (ALP) activity, and both functions can be increased by 100-200% over that of pristine Ti substrates while retaining high antibacterial efficacy. Such substrates can be expected to have good potential in orthopedic applications.     

Gold nanoparticles functionalized with therapeutic and targeted peptides for cancer treatment

Functionalization of nanostructures such as gold nanoparticles (AuNPs) with different biological molecules has many applications in biomedical imaging, clinical diagnosis and therapy. Researchers mostly employed AuNPs larger than 10 nm for different biological and medicinal applications in previous studies. Herein, we synthesized a novel small (2 nm) AuNPs, which were functionalized with the therapeutic peptide, PMI (p12), and a targeted peptide, CRGDK for selective binding to neuropilin-1(Nrp-1) receptors which overexpressed on the cancer cells and regulated the process of membrane receptor-mediated internalization. It was found that CRGDK peptides increased intracellular uptake of AuNPs compared to other surface conjugations quantified by ICP-MS. Interestingly, CRGDK functionalized AuNPs resulted in maximal binding interaction between the CRGDK peptide and targeted Nrp-1 receptor overexpressed on MDA-MB-321 cell surface, which improved the delivery of therapeutic P12 peptide inside targeted cells. Au@p12 + CRGDK nanoparticles indicated with highly effective cancer treatment by increasing p53 expression upregulated with intracellular enhanced p12 therapeutic peptide. These results have implications to design and functionalize different molecules onto AuNPs surfaces to make hybrid model system for selective target binding as well as therapeutic effects for cancer treatment.     

Recombinant spider silk with cell binding motifs for specific adherence of cells

Silk matrices have previously been shown to possess general properties governing cell viability. However, many cell types also require specific adhesion sites for successful in vitro culture. Herein, we have shown that cell binding motifs can be genetically fused to a partial spider silk protein, 4RepCT, without affecting its ability to self-assemble into stable matrices directly in a physiological-like buffer. The incorporated motifs were exposed in the formed matrices, and available for binding of integrins. Four different human primary cell types; fibroblasts, keratinocytes, endothelial cells and Schwann cells, were applied to the matrices and investigated under serum-free culture conditions. Silk matrices with cell binding motifs, especially RGD, were shown to promote early adherence of cells, which formed stress fibers and distinct focal adhesion points. Schwann cells acquired most spread-out morphology on silk matrices with IKVAV, where significantly more viable cells were found, also when compared to wells coated with laminin. This strategy is thus suitable for development of matrices that allow screening of various cell binding motifs and their effect on different cell types.     

Focal adhesion kinase functions as a receptor-proximal signaling component required for directed cell migration

In performing host-defense functions, cells of the immune system become activated by soluble chemokine signals and must migrate through endothelial cell or solid tissue barriers to reach sites of inflammation or infection. Regulated adhesive interactions of immune cells with endothelium, extracellular matrix components, and cells of solid organs are critical control points of the overall immune response. Both the soluble chemokine and cell adhesion receptor-mediated migration signals must converge on common intracellular targets to engage the cell migration machinery. In this article, we focus on the role of focal adhesion kinase (FAK) and its homolog Pyk2 as cytoplasmic mediators of motility events in multiple cell types. We introduce the overall domain structure of the FAK and Pyk2 nonreceptor protein tyrosine kinases (PTKs), high-light some of the signals that activate these PTKs, and detail the molecules that functionally interact and signal transduction pathways that may mediate cell migration responses. Emphasis is placed on the knowledge gained from studies using FAK-null cells as a model system to decipher the role of this PTK in promoting cell motility.     

A DNA fluorometric assay for measuring fish cell proliferation in microplates with different well sizes

Summary The use of a DNA-binding dye, bisbenzimidazole (H33258), and a microplate fluorometer, CytoFluor 2350, was optimized to measure cell number in Chinook salmon embryo cell cultures (CHSE-214). The uniformity of cell homogenates, which were prepared prior to staining, was evaluated by area scan, which consists of multiple measurements over the total well area. Disruption in 0.01% SDS produced a relatively uniform homogenate and a low background. Homogenates were stained with H33258 at 1 to 10 µg/ml. Linear relationships between cell numbers and fluorescence units were established in 96 well plates, which were read only by standard scan, and in 6, 12, 24 and 48 well plates, which were read by area and standard scan. Area scan provided better relationships. These methods were used to show that in L-15 medium CHSE-214 were able to attach, retain viability, and proliferate with very little exogenous calcium.     

Intercellular adhesion molecule-1 (ICAM-1) in cervicovaginal fluid of women presenting with preterm labor: predictive value for preterm delivery

PROBLEM: Clinically useful tests for the prediction and diagnosis of preterm labor and delivery remain to be established. We have hypothesized that soluble intercellular adhesion molecule-1 (sICAM-1) in the cervicovaginal fluid of women with preterm labor may be a useful diagnostic tool. METHOD OF STUDY: The cervicovaginal fluid of 103 women between 24(0) and 33(6) weeks gestation with preterm contractions and intact membranes was assayed for sICAM-1. RESULTS: Elevated sICAM-1 concentrations predicted short intervals to delivery (area under receiver operator characteristic (ROC) curves, 0.70-0.72 for delivery within 3, 7 and 10 days), with high specificity. Characteristics for delivery within 3 days at a 3 ng/mL threshold for a positive test were sensitivity 33.3%, specificity 98.9%, and positive and negative predictive values of 75.0% and 93.9%, respectively. Predictive ability was independent of and complementary to that of fetal fibronectin (fFN). CONCLUSIONS: Measurement of sICAM-1 in cervicovaginal fluid has potential as a predictor of preterm delivery in women with symptoms of preterm labor, particularly in conjunction with fFN testing.     

Multifunctional polymer scaffolds with adjustable pore size and chemoattractant gradients for studying cell matrix invasion

Transmigrating cells often need to deform cell body and nucleus to pass through micrometer-sized pores in extracellular matrix scaffolds. Furthermore, chemoattractive signals typically guide transmigration, but the precise interplay between mechanical constraints and signaling mechanisms during 3D matrix invasion is incompletely understood and may differ between cell types. Here, we used Direct Laser Writing to fabricate 3D cell culture scaffolds with adjustable pore sizes (2–10 μm) on a microporous carrier membrane for applying diffusible chemical gradients. Mouse embryonic fibroblasts invade 10 μm pore scaffolds even in absence of chemoattractant, but invasion is significantly enhanced by knockout of lamin A/C, a known regulator of cell nucleus stiffness. Nuclear stiffness thus constitutes a major obstacle to matrix invasion for fibroblasts, but chemotaxis signals are not essential. In contrast, epithelial A549 cells do not enter 10 μm pores even when lamin A/C levels are reduced, but readily enter scaffolds with pores down to 7 μm in presence of chemoattractant (serum). Nuclear stiffness is therefore not a prime regulator of matrix invasion in epithelial cells, which instead require chemoattractive signals. Microstructured scaffolds with adjustable pore size and diffusible chemical gradients are thus a valuable tool to dissect cell-type specific mechanical and signaling aspects during matrix invasion.     

Correlation of a dynamic model for immunological synapse formation with effector functions: two pathways to synapse formation

During antigen recognition by T cells different receptors and ligands form a pattern in the intercellular junction called the immunological synapse, which might be involved in T-cell activation. Recently, a synapse assembly model has been proposed, which enables the calculation of the propensity for synapse assembly driven by membrane-constrained protein binding interactions. We bring together model predictions of mature synapse assembly with data on the dependence of T-cell responses on T-cell receptor (TCR)-MHC-peptide (pMHC) binding kinetics. Predictions of mature synapse assembly, based on TCR-pMHC binding kinetics, correlate well with observed cytokine responses by T cells bearing the relevant TCR but not with cytotoxic T lymphocyte-mediated killing. We discuss the suggested different role for the synapse in pre- and post-nuclear activation events in T cells. The view of immunological synapse assembly given here emphasizes the importance of both the on and off rates for the TCR-pMHC interaction and in this context recent data on a positive role for analogs of self-peptides in synapse assembly is considered.     

Expansion of T cells negative for CD28 expression in hiv infection. Relation to activation markers and cell adhesion molecules,and correlation with prognostic markers

  CD28 is a transmembrane glycoprotein that provides T cells with an essential co-stimulatory signal during antigen presentation. Using flow cytometry, we document here an expansion of CD28^– T cells in HIV infection. Whereas the percentage of CD4⁺CD28⁺ T cells among total lymphocytes was decreased, a small increase of the percentage of CD4⁺CD28^– T cells was observed. In the CD8⁺ subset, there was a marked expansion of CD8⁺CD28^– T cells. An increased percentage of CD8⁺ T cells positive for HLA-DR was found in both CD28⁺ and CD28^– cells. Results were similar for CD38 expression. HIV infection was also distinguished by a shift from LFA-1^lowCD28^low to LFA-1^highCD28^high and LFA-1^high-CD28^neg expression pattern on CD8⁺ T cells. Negative correlations were found between percentage and absolute number of CD8⁺CD28⁺ T cells and several serum parameters usually associated with poor prognosis (IgA, IgE, β2-microglobulin and HIV-1 p24 antigen). Thus, HIV infection is characterized by a marked expansion of CD28^– T cells with an abnormal expression of activation markers and cell adhesion molecules. In addition, CD8⁺CD28⁺, but not CD8⁺CD28^– or total CD8⁺ T cell numbers, correlated with the levels of established serological markers of disease severity or progression and may, therefore, have predictive value. Received: 1 November 1995